Tunable Polymer-Graphene Oxide Composite for Single Cell Analysis of Breast Cancer CTCs Traditionally, tissue biopsies are used for molecular characterization of tumor, both at initial diagnosis and at the time of recurrence. However, multiple biopsies are not only expensive but also painful and may not always be feasible. The concept of blood biopsy/liquid biopsy is garnering momentum not only due to a non-invasive nature, but also due to the feasibility of frequent monitoring of patients to track response and resistance. Circulating tumor cells (CTCs) shed from primary tumor carry the key molecular signatures and could act as ideal surrogates for tissue biopsies. Recent advances in isolation technologies combined with the next generation tools for molecular analysis have shifted the paradigm in favor of liquid biopsy. Nanomaterials offer excellent prospects to improve the sensitivity of biomolecule detection because they have a high surface area to volume ratio and are similar in size to biomolecules. Recently, nanostructures such as silicon nanopillars, quartz nanowires, and TiO2 nanofibers are being used for CTC capture. These studies showed enhanced CTC capture efficiency. However, the capture yields, and throughput were found to be lower than those observed on microfluidic-based CTC chips. Our laboratory has been working on developing microfluidic technologies that can isolate CTCs with high sensitivity and specificity. We have developed a new, highly sensitive technology GO Chip to effectively isolate CTCs, incorporating a nanomaterial graphene oxide (GO). GO is a promising nanomaterial with emerging applications such as water-insoluble cancer drug delivery, serving as a medium for biosensors for bacterial assays and DNA detectio, paper-like material, and polymer composites. The advantages of GO include: 1) high surface to volume ratio 2) ease of immobilizing functional groups using PEG based chemistry, 3) precise size-control by sonication time and filtration, and 4) promising optical properties for biological and medical research. The immobilization of nanomaterial, GO on microfluidic devices enhances the surface area many fold. Self-assembly of GO creates islands of nano-arms for sensitive CTC capture without the aid of three-dimensional structures. We have demonstrated the isolation of cells with high sensitivity even at low frequency of target cells (>95 percent). Although immunoaffinity based approaches are sensitive and yield pure population of CTCs compared to label free technologies, they suffer from low throughput and the attachment of CTCs to the functionalized substrates. In this proposed work we aim to develop a tunable thermal sensitive Grapheme Oxide polymer complex along with a radial flow strategy to isolate CTCs at 10mL/hr using a GO-Polymer composite material. Once the CTCs are isolated, the polymer can be melted by simply lowering the temperature to 15C. The overreaching goal is to capture CTCs from breast cancer patients and molecularly characterize them using novel single cell analysis tools to identify the stem signature. Screening and improved adjuvant treatments have increased breast cancer survival rates since the mid- 1970s, with current 5 year survival rates at nearly 90 percent. Despite these significant improvements, the chances of recurrent or relapsed disease are sobering. Recent studies have suggested that cancer stem cells (CSCs) are immortal tumor-initiating cells that can self-renew and have pluripotent capacity. CSCs have been identified in multiple malignancies, including leukemia and various solid cancers. Due to their extraordinary characteristics, CSCs are thought to be the basis for tumor initiation, development, metastasis recurrence and drug resistance. There has been significant progress in elucidating the pathways which regulate CSCs. As a result, agents developed to target CSC self-renewal and survival pathways have been identified and early stage clinical trials utilizing CSC inhibitors are in progress. Given the clinical importance of CSCs and the need to monitor these cells for patients on clinical trials, there is an urgency to develop methodology to assay these cell populations in patients with cancer. CTCs carry the signature of the primary tumor, have the ability to form new tumors and hence play a central role in lethal progression of disease. Currently, enumeration of CTCs is associated with prognosis in breast cancer; however, less is known about the clinical relevance of their molecular characterization, offering a promising topic in cancer research to improve cancer outcomes and individualized treatments. We hypothesize that CTCs will be highly enriched for CSCs, and analyzing this subpopulation will reveal the markers of recurrence, metastasis and a way to monitor the effectiveness of CSC targeted therapies. Our rigorous preliminary studies, combined with the excellence of our research team and environment, underscore the high likelihood for success. We expect outcomes of this research to ultimately impact both diagnosis and therapy of breast cancer resulting in greater numbers of patients cured of this disease. The overreaching challenges that we would like to address are why certain cancers recur and eventually becomes life threatening. Importantly, identifying the signature of aggressive cells in circulation may provide useful prognostic and predictive information to guide treatment decisions.